Revisiting the TLC analysis of commercial Sceletium products with a revised technique Early visualisation of mesembrine-type alkaloids via TLC was plagued by the use of crude iodine vapor visualisation which was non-specific, making accurate detection of alkaloids problematic. As noted previously, there are several distinct Sceletium chemotypes which makes having a specific reference material a challenge, coupled with extremely variable alkaloid levels in the raw plant material. To improve the visualisation technique, Dragendorff's reagent was selected as an alkaloid- selective method. This uses a potassium bismuth iodide complex which forms with (mainly tertiary nitrogenous) alkaloids to form a yellow-red-orange-brown colouration [1]. Failure to properly visualise the relevant compounds also made developing a mobile phase for TLC problematic. Initial attempts relied on a single solvent system which was likely not optimised. This experiment aimed to revisit the solvent system to find a better option. An example of a Dragendorff visualised TLC plate is shown below: The formation of reagent is generally described as follows: The bismuth ions from bismuth subnitrate react with potassium iodide and form at first the black precipitate of bismuth iodide 1 Bi 3+ + 3KI → BiI 3 + 3K + Then, after the complete sedimentation of bismuth(III)iodide, the excess of iodide ions reacts to latter to form an orange colored, soluble complex of potassiumtetraiodobismuthate. Bi 3+ + KI → K[BiI 4 ] Then , an ion - exchange reaction takes place between ammonium salt and potassiumtetraiodo bismuthate leading to the formation of an insoluble complex salt [ R 3 NH + X - ] + K [ BiI 4 ] → [ R 3 NH ]+[ BiI 4 ] - + KX Depending on the nature of alkaloid ( or tertiary amine ), this ion pair has a yellow to orange to red to brown color Secondary amines will create less intensive colors An example of the mesembrine-tetraiodo bismuthate complex A second stain used was a modified vanillin stain, which is reactive for strong and weak nucleophiles (alcohols, amines), and for many aldehydes and ketones 2 An example of a vanillin stained TLC plate A reference sample of verified Sceletium tortuosum was collected from verified plant material and prepared as both unfermented and fermented dry powders. These were compared to a commercial raw herb, 100:1 and standardised @ 2% mesembrine product. According to the lierature, alkaloid concentrations in the raw plant material can vary considerably. The reference material was collected from this S. tortuosum plant. 3 Two Reference samples showing the red-orange fluorescence 4 Another extract (100:1) showing the green-yellow fluorescence Preparation of Dragendorff's: Dissolve 10g of tartaric acid in 40mL of water, add 0.85g bismuth subnitrate and shake for one hour. Add 20mL of water containing 8g KI and shake vigorously. Stand for 24 hrs and filter. Store in a place protected from light. Modified Preparation of Dragendorff's via readily available OTC bismuth subsalicylate If you're unable to obtain bismuth subnitrate, you can use the following alternative procedure to make up a usable solution of Dragendorff's Reagent from Pepto-Bismol tablets or the generic equivalent. These tablets contain 262 mg of bismuth subsalicylate per tablet, which is equivalent in bismuth mass to about 212 mg of bismuth subnitrate. We can therefore substitute two Pepto-Bismol tablets for the 0.4 g of bismuth subnitrate. This alternative method isn't pretty, but it does use readily available materials and it does work. 1. Transfer two Pepto-Bismol tablets and about 20 mL of water to a beaker. 2. Swirl the beaker until the tablets have broken up into powder. Bismuth subsalicylate and the binders used in the tablets are both very insoluble in water, so don't be concerned if it looks as though none of the powder has dissolved. 3. Add about 10 mL of concentrated hydrochloric acid to the beaker. Swirl the beaker occasionally until foaming ceases. At this point, the liquid appears chalky white. 4. Allow the contents of the beaker to settle. Most of the solid matter precipitates, but enough remains suspended to give the liquid a cloudy white appearance. 5. Filter or carefully decant the liquid into a second beaker to remove as much as possible of the undissolved solids. 5 6. Dissolve about 7.0 g of potassium iodide in a few mL of water and transfer that solution to the beaker that contains the bismuth solution. The solution immediately assumes a yellow-brown, cloudy appearance. 7. Bring up the volume in the beaker to about 100 mL with distilled water, allow the solid material to settle, and then pour off the clear yellow-brown solution into a storage bottle labeled Dragendorff Reagent. Preparation of modified vanillin stain 5g of vanillin was dissolved in 100mL of 95% ethanol and 1mL conc. 85% phosphoric acid added. Sample preparation: 500mg of the sample was macerated in 2mL 95% ethanol and basified with 0.25mL aq. ammonia. The ethanolic solution was spotted on the TLC plate. Results Interestingly, the Reference sample solutions of verified Sceletium tortuosum material, fermented and unfermented gave a vivid orange-red fluorescence under UV, while others gave a light yellow flourescence. From TLC with acetone:white spirits 1:1 these constituents seemed to be high Rf, slightly visible pigments. Initial TLC was tried with acetone and 3:1 acetone:white spirits, the latter separating the fluorescent bands distinctly but with trailing of the lower Rf constituents. 3:1:1 acetone:white spirits: 95% ethanol gave some visible separation of lower Rf constituents (I 2 visualisation). Vanillin staining was even less remarkable, with some blue-purple high Rf constituents and pale orange lower Rf constituents. 6 3:1:1 Acetone:EtOH:white spirits TLC, I 2 3:1:1 Acetone:EtOH:white spirits, vanillin stain To verify the validity of Dragendorff's for visualisation, spot tests were conducted on each sample to test for the presence of alkaloids. This was done by spotting a small aliquot of each sample onto the plate and allowing to dry, then spraying with the reagent. Remarkably, only one Sceletium product gave a strong positive (top left): 7 This positive sample was claimed to be 2.2% total alkaloids. Others were only very weakly positive - including the selected reference materials. This may explain why TLC has been so challenging, as only one sample is alkaloid rich according to the reagent. Following up with TLC of the samples that gave weak positives was generally without results, the alkaloids failing to visualise. TLC of the sample that did test alkaloid positive, above, was more remarkable, with distinct separation of a major high Rf =0.82 constituent and lesser minor constituents (Rf = 0.66, 0.39 and 0.26). Conclusions: The more selective visualisation reagent Dragendorff's revealed that the majority of 8 Sceletium products were not alkaloid rich. It may be possible something was masking the reagent in other samples. This poses an interesting question of how many actually contain active alkaloids? References Raal A, Meos A, Hinrikus T, Heinämäki J, Romāne E, Gudienė V, Jak Tas V, Koshovyi O, Kovaleva A, Fursenco C, Chiru T, Nguyen HT. Dragendorff's reagent: Historical perspectives and current status of a versatile reagent introduced over 150 years ago at the University of Dorpat, Tartu, Estonia. Pharmazie. 2020 Jul 1;75(7):299-306. doi: 10.1691/ph.2020.0438 Sceletium Chemotypes https://pdfhost.io/v/Y0Inx35hj6_sceletium_chemotypes Kowalska, T.; Sajewicz, M. Thin-Layer Chromatography (TLC) in the Screening of Botanicals– Its Versatile Potential and Selected Applications. Molecules 2022, 27, 6607. https://doi.org/10.3390/molecules27196607 Visualising TLC plates https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab_Tech niques_(Nichols)/02%3A_Chromatography/2.03% 3A_Thin_Layer_Chromatography_(TLC)/2.3F%3A_Visualizing_TLC_Plates 9